Ebook Microbiological applications: Laboratory manual in general microbiology: Part 2

(BQ) Part 2 book Microbiological applications: Laboratory manual in general microbiology presents the following contents: Microbiology of water, microbiology of milk and food products, medical microbiology and immunology. Invite you to consult.

Applications Lab Manual,

wa-The threat to human welfare by contamination of water supplieswith sewage is a prime concern of everyone The enteric diseasessuch as cholera, typhoid fever, and bacillary dysentery often result

in epidemics when water supplies are not properly protected ortreated Thus, our prime concern in this unit is the sanitary phase

of water microbiology The American Public Health Association in

its Standard Methods for the Examination of Water and Wastewater

has outlined acceptable procedures for testing water for sewagecontamination The exercises of this unit are based on the proce-dures in that book

11

Applications Lab Manual,

Water that contains large numbers of bacteria may be

perfectly safe to drink The important consideration,

from a microbiological standpoint, is the kinds of

mi-croorganisms that are present Water from streams

and lakes that contain multitudes of autotrophs and

saprophytic heterotrophs is potable as long as

pathogens for humans are lacking The intestinal

pathogens such as those that cause typhoid fever,

cholera, and bacillary dysentery are of prime concern

The fact that human fecal material is carried away by

water in sewage systems that often empty into rivers

and lakes presents a colossal sanitary problem; thus,

constant testing of municipal water supplies for the

presence of fecal microorganisms is essential for the

maintenance of water purity

Routine examination of water for the presence of

intestinal pathogens would be a tedious and difficult,

if not impossible, task It is much easier to

demon-strate the presence of some nonpathogenic intestinal

types such as Escherichia coli or Streptococcus

fae-calis Since these organisms are always found in the

intestines, and normally are not present in soil or

wa-ter, it can be assumed that their presence in water

in-dicates that fecal material has contaminated the water

supply

E coli and S faecalis are classified as good

sewage indicators The characteristics that make

them good indicators of fecal contamination are (1)

they are normally not present in water or soil, (2) they

are relatively easy to identify, and (3) they survive a

little longer in water than enteric pathogens If they

were hardy organisms, surviving a long time in water,

they would make any water purity test too sensitive

Since both organisms are non-spore-formers, their

survival in water is not extensive

E coli and S faecalis are completely different

organisms E coli is a gram-negative

non-spore-forming rod; S faecalis is a gram-positive coccus.

The former is classified as a coliform; the latter is an

enterococcus Physiologically, they are also

com-pletely different

The series of tests depicted in figure 63.1 is based

Since S faecalis is not a coliform, a completely

differ-ent set of tests must be used for it

Note that three different tests are shown in figure63.1: presumptive, confirmed, and completed Eachtest exploits one or more of the characteristics of a co-liform A description of each test follows

Presumptive Test In the presumptive test a series

of 9 or 12 tubes of lactose broth are inoculated withmeasured amounts of water to see if the water con-tains any lactose-fermenting bacteria that producegas If, after incubation, gas is seen in any of the lac-

tose broths, it is presumed that coliforms are present

in the water sample This test is also used to determinethe most probable number (MPN) of coliforms pres-ent per 100 ml of water

Confirmed Test In this test, plates of Levine EMBagar or Endo agar are inoculated from positive (gas-producing) tubes to see if the organisms that areproducing the gas are gram-negative (another co-liform characteristic) Both of these media inhibitthe growth of gram-positive bacteria and causecolonies of coliforms to be distinguishable fromnoncoliforms On EMB agar coliforms producesmall colonies with dark centers (nucleatedcolonies) On Endo agar coliforms produce reddishcolonies The presence of coliform-like coloniesconfirms the presence of a lactose-fermentinggram-negative bacterium

Completed Test In the completed test our concern

is to determine if the isolate from the agar plates trulymatches our definition of a coliform Our media forthis test include a nutrient agar slant and a Durhamtube of lactose broth If gas is produced in the lactosetube and a slide from the agar slant reveals that wehave a gram-negative non-spore-forming rod, we can

be certain that we have a coliform

The completion of these three tests with tive results establishes that coliforms are present;

posi-Applications Lab Manual,

Eighth Edition

Examination of Water:

Qualitative Tests

Companies, 2001

Bacteriological Examination of Water: Qualitative Tests • Exercise 63

Figure 63.1 Bacteriological analysis of water

Applications Lab Manual,

In this exercise, water will be tested from local

ponds, streams, swimming pools, and other sources

supplied by students and instructor Enough known

positive samples will be evenly distributed

through-out the laboratory so that all students will be able to

see positive test results All three tests in figure 63.1

will be performed If time permits, the IMViC tests

may also be performed

As stated earlier, the presumptive test is used to

de-termine if gas-producing lactose fermenters are

pres-ent in a water sample If clear surface water is being

tested, nine tubes of lactose broth will be used as

shown in figure 63.1 For turbid surface water an

ad-ditional three tubes of single strength lactose broth

will be inoculated

In addition to determining the presence or

ab-sence of coliforms, we can also use this series of

lac-tose broth tubes to determine the most probable

number (MPN) of coliforms present in 100 ml of

water A table for determining this value from the

number of positive lactose tubes is provided in

Appendix A

Before setting up your test, determine whether

your water sample is clear or turbid Note that a

sep-arate set of instructions is provided for each type of

water

Clear Surface Water

If the water sample is relatively clear, proceed as

Note: DSLB designates double strength lactose

broth It contains twice as much lactose as

SSLB (single strength lactose broth)

1 Set up 3 DSLB and 6 SSLB tubes as illustrated in

figure 63.1 Label each tube according to the

amount of water that is to be dispensed to it: 10

ml, 1.0 ml, and 0.1 ml, respectively.

2 Mix the bottle of water to be tested by shaking 25

times

3 With a 10 ml pipette, transfer 10 ml of water to

5 Incubate the tubes at 35° C for 24 hours

6 Examine the tubes and record the number of tubes

in each set that have 10% gas or more

7 Determine the MPN by referring to table VI,Appendix A Consider the following:

Example: If you had gas in the first three tubes

and gas only in one tube of the second series, butnone in the last three tubes, your test would beread as 3–1–0 Table VI indicates that the MPNfor this reading would be 43 This means that thisparticular sample of water would have approxi-mately 43 organisms per 100 ml with 95% prob-ability of there being between 7 and 210 organ-

isms Keep in mind that the MPN figure of 43 is

only a statistical probability figure.

8 Record the data on the Laboratory Report

Turbid Surface Water

If your water sample appears to have considerablepollution, do as follows:

1 water blank (99 ml of sterile water)

Note: See comment in previous materials list

0.1 ml, and the last three tubes 0.01 ml.

3 Mix the bottle of water to be tested by shaking

7 With a fresh 1 ml pipette, transfer 1.0 ml of water

from the blank to the remaining tubes of SSLB.This is equivalent to adding 0.01 ml of full-strength water sample

8 Incubate the tubes at 35° C for 24 hours

9 Examine the tubes and record the number of tubes

Exercise 63 • Bacteriological Examination of Water: Qualitative Tests

Applications Lab Manual,

Eighth Edition

Examination of Water:

Qualitative Tests

Companies, 2001

a Select the three consecutive sets of tubes that

have at least one tube with no gas

b If the first set of tubes (10 ml tubes) are not

used, multiply the MPN by 10

Example: Your tube reading was 3–3–3–1 What

is the MPN?

The first set of tubes (10 ml) is ignored and

the figures 3–3–1 are applied to the table The

MPN for this series is 460 Multiplying this by 10,

the MPN becomes 4600

Example: Your tube reading was 3–1–2–0 What

is the MPN?

The first three numbers are (3–1–2) applied to

the table The MPN is 210 Since the last set of

tubes is ignored, 210 is the MPN

Once it has been established that gas-producing

lac-tose fermenters are present in the water, it is presumed

to be unsafe However, gas formation may be due to

noncoliform bacteria Some of these organisms, such

as Clostridium perfringens, are gram-positive To

confirm the presence of gram-negative lactose

fer-menters, the next step is to inoculate media such as

Levine eosin–methylene blue agar or Endo agar from

positive presumptive tubes

Levine EMB agar contains methylene blue,

which inhibits gram-positive bacteria Gram-negative

lactose fermenters (coliforms) that grow on this

medium will produce “nucleated colonies” (dark

cen-ters) Colonies of E coli and E aerogenes can be

dif-ferentiated on the basis of size and the presence of a

greenish metallic sheen E coli colonies on this

medium are small and have this metallic sheen,

whereas E aerogenes colonies usually lack the sheen

and are larger Differentiation in this manner is not

completely reliable, however It should be

remem-bered that E coli is the more reliable sewage

indica-tor since it is not normally present in soil, while E.

aerogenes has been isolated from soil and grains.

Endo agar contains a fuchsin sulfite indicator

that makes identification of lactose fermenters

rela-tively easy Coliform colonies and the surrounding

medium appear red on Endo agar Nonfermenters of

lactose, on the other hand, are colorless and do not

af-fect the color of the medium

In addition to these two media, there are several

other media that can be used for the confirmed test

Brilliant green bile lactose broth, Eijkman’s medium,

and EC medium are just a few examples that can beused

To demonstrate the confirmation of a positivepresumptive in this exercise, the class will use LevineEMB agar and Endo agar One half of the class willuse one medium; the other half will use the othermedium Plates will be exchanged for comparisons

2 Incubate the plate for 24 hours at 35° C

3 Look for typical coliform colonies on both kinds

of media Record your results on the LaboratoryReport If no coliform colonies are present, thewater is considered bacteriologically safe todrink

Note: In actual practice, confirmation of all

pre-sumptive tubes would be necessary to ensure curacy of results

A final check of the colonies that appear on the firmatory media is made by inoculating a nutrientagar slant and a Durham tube of lactose broth Afterincubation for 24 hours at 35° C, the lactose broth isexamined for gas production A gram-stained slide ismade from the slant, and the slide is examined underoil immersion optics

con-If the organism proves to be a gram-negative,non-spore-forming rod that ferments lactose, weknow that coliforms were present in the tested watersample If time permits, complete these last tests andrecord the results on the Laboratory Report

THEIMViC TESTS

Review the discussion of the IMViC tests on page

175 The significance of these tests should be muchmore apparent at this time Your instructor will indi-cate whether these tests should also be performed ifyou have a positive completed test

Bacteriological Examination of Water: Qualitative Tests • Exercise 63

Applications Lab Manual,

Eighth Edition

The Membrane Filter Method

64

In addition to the multiple tube test, a method utilizing

the membrane filter has been recognized by the United

States Public Health Service as a reliable method for

the detection of coliforms in water These filter disks

are 150 micrometers thick, have pores of 0.45

microm-eter diammicrom-eter, and have 80% area perforation The

pre-cision of manufacture is such that bacteria larger than

0.47 micrometer cannot pass through Eighty percent

area perforation facilitates rapid filtration

To test a sample of water, the water is passed

through one of these filters All bacteria present in the

sample will be retained directly on the filter’s surface

The membrane filter is then placed on an absorbent

pad saturated with liquid nutrient medium and

incu-bated for 22 to 24 hours The organisms on the filter

disk will form colonies that can be counted under the

microscope If a differential medium such as m Endo

MF broth is used, coliforms will exhibit a

characteris-tic golden metallic sheen

The advantages of this method over the multiple

tube test are (1) higher degree of reproducibility of

re-sults; (2) greater sensitivity since larger volumes of

water can be used; and (3) shorter time (one-fourth)

for getting results

Figure 64.1 illustrates the procedure we will use

in this experiment

Materials:

vacuum pump or water faucet aspirators

membrane filter assemblies (sterile)

side-arm flask, 1000 ml size, and rubber hose

sterile graduates (100 ml or 250 ml size)

sterile, plastic Petri dishes, 50 mm dia

1 Prepare a small plastic Petri dish as follows:

a With a flamed forceps, transfer a sterile sorbent pad to a sterile plastic Petri dish

ab-b Using a 5 ml pipette, transfer 2.0 ml of m Endo

MF broth to the absorbent pad

2 Assemble a membrane filtering unit as follows:

a Aseptically insert the filter holder base into the

neck of a 1-liter side-arm flask

b With a flamed forceps, place a sterile brane filter disk, grid side up, on the filterholder base

mem-c Place the filter funnel on top of the membranefilter disk and secure it to the base with theclamp

3 Attach the rubber hose to a vacuum source (pump

or water aspirator) and pour the appropriateamount of water into the funnel

The amount of water used will depend on ter quality No less than 50 ml should be used.Waters with few bacteria and low turbidity permitsamples of 200 ml or more Your instructor willadvise you as to the amount of water that youshould use Use a sterile graduate for measuringthe water

wa-4 Rinse the inner sides of the funnel with 20 ml ofsterile water

5 Disconnect the vacuum source, remove the nel, and carefully transfer the filter disk with ster-

fun-ile forceps to the Petri dish of m Endo MF broth.

Keep grid side up.

6 Incubate at 35° C for 22 to 24 hours Don’t

Applications Lab Manual,

Eighth Edition

The Membrane Filter Method • Exercise 64

Figure 64.1 Membrane filter routine

Applications Lab Manual,

Eighth Edition

Standard Plate Count:

A Quantitative Test

65

In determining the total numbers of bacteria in

wa-ter, we are faced with the same problems that are

en-countered with soil Water organisms have great

variability in physiological needs, and no single

medium, pH, or temperature is ideal for all types

Despite the fact that only small numbers of

organ-isms in water will grow on nutrient media, the

stan-dard plate count can perform an important function

in water testing Probably its most important use is

to give us a tool to reveal the effectiveness of

vari-ous stages in the purification of water Plate counts

made of water before and after storage, for example,

can tell us how effective holding is in reducing

bac-terial numbers

In this exercise, various samples of water will be

evaluated by routine standard plate count

proce-dures Since different dilution procedures are

re-quired for different types of water, two methods are

given

If the water is of low bacterial count, such as in the

case of tap water, use the following method

Materials:

1.0 ml pipettes

2 tryptone glucose extract agar pours (TGEA)

2 sterile Petri plates

Quebec colony counter and hand counters

water samples

1 Liquefy two tubes of TGEA and cool to 45° C

2 After shaking the sample of water 25 times

trans-fer 1 ml of water to each of the two sterile Petri

plates

3 Pour the medium into the dishes, rotate ciently to get good mixing of medium and water,and let cool

suffi-4 Incubate at 35° C for 24 hours

5 Count the colonies of both plates on the Quebeccolony counter and record your average count ofthe two plates on the Laboratory Report

If the water is likely to have a high bacterial count, as

in the case of surface water, proceed as follows:

1 Liquefy a bottle of TGEA medium and cool to45° C

2 After shaking your water sample 25 times, duce two water blanks with dilutions of 1:100 and1:1000 See Exercise 23

pro-3 Distribute aliquots from these blanks to six Petridishes, which will provide you with two plateseach of 1:100, 1:1000, and 1:10,000 dilutions

4 Pour one-sixth of the TGEA medium into eachplate and rotate sufficiently to get even mixing ofthe water and medium

5 Incubate at 35° C for 24 hours

6 Select the pair of plates that has 30 to 300colonies on each plate and count all the colonies

on both plates Record the average count for thetwo plates on the second portion of LaboratoryReport 64, 65

Applications Lab Manual,

Eighth Edition

Microbiology of Milk and Food Products

Milk and food provide excellent growth media for bacteria whensuitable temperatures exist This is in direct contrast to natural wa-ters, which lack the essential nutrients for pathogens The intro-duction of a few pathogens into food or milk products becomes amuch more serious problem because of the ability of these sub-stances to support tremendous increases in bacterial numbers.Many milk-borne epidemics of human diseases have been spread

by contamination of milk by soiled hands of dairy workers, itary utensils, flies, and polluted water supplies The same thing can

unsan-be said for improper handling of foods in the home, restaurants,hospitals, and other institutions

We learned in Part 11 that bacteriological testing of water is marily qualitative—emphasis being placed on the presence or ab-sence of coliforms as indicators of sewage Bacteriological testing

pri-of milk and food may also be performed in this same manner, ing similar media and procedures to detect the presence of coli-forms However, most testing by public health authorities is quan-titative Although the presence of small numbers of bacteria inthese substances does not necessarily mean that pathogens arelacking, low counts do reflect better care in handling of food andmilk than is true when high counts are present

us-Standardized testing procedures for milk products are outlined

by the American Public Health Association in Standard Methods for

the Examination of Dairy Products The procedures in Exercises 66,

67, and 67 are excerpts from that publication Copies of the bookmay be available in the laboratory as well as in the library

Exercises 69, 70, and 71 pertain to bacterial counts in dried fruitand meats, as well as to spoilage of canned vegetables and meats.Since bacterial counts in foods are performed with some of thetechniques you have learned in previous exercises, you will have anopportunity to apply some of those skills here Exercises 72 and 73pertain to fermentation methods used in the production of wine andyogurt

12

Applications Lab Manual,

Eighth Edition

Standard Plate Count of Milk

66

The bacterial count in milk is the most reliable

indi-cation we have of its sanitary quality It is for this

rea-son that the American Public Health Association

rec-ognizes the standard plate count as the official method

in its Milk Ordinance and Code Although human

pathogens may not be present in a high count, it may

indicate a diseased udder, unsanitary handling of

milk, or unfavorable storage temperatures In general,

therefore, a high count means that there is a greater

likelihood of disease transmission On the other hand,

it is necessary to avoid the wrong interpretation of low

plate counts, since it is possible to have pathogens

such as the brucellosis and tuberculosis organisms

when counts are within acceptable numbers Routine

examination and testing of animals act as safeguards

against the latter situation

In this exercise, standard plate counts will be made

of two samples of milk: a supposedly good sample and

one of known poor quality Odd-numbered students will

work with the high-quality milk and even-numbered

stu-dents will test the poor-quality sample A modification

of the procedures in Exercise 23 will be used

Materials:

milk sample

1 sterile water blank (99 ml)

4 sterile Petri plates

1.1 ml dilution pipettes

1 bottle of TGEA (40 ml)

Quebec colony counter

mechanical hand counter

1 Following the procedures used in Exercise 23,pour four plates with dilutions of 1:1, 1:10, 1:100,and 1:1000 Before starting the dilution proce-dures, shake the milk sample 25 times in the cus-tomary manner

2 Incubate the plates at 35° C for 24 hours andcount the colonies on the plate that has between

3 sterile water blanks (99 ml)

4 sterile Petri plates1.1 ml dilution pipettes

1 bottle TGEA (50 ml)Quebec colony countermechanical hand counter

1 Following the procedures used in Exercise 23,pour four plates with dilutions of 1:10,000,1:100,000, 1:1,000,000, and 1:10,000,000 Beforestarting the dilutions, shake the milk sample 25times in the customary manner

2 Incubate the plates at 35° C for 24 hours andcount the colonies on the plate that has between

30 and 300 colonies

3 Record your results on the first portion ofLaboratory Report 66, 67

Applications Lab Manual,

Eighth Edition

and Food Products Count of Organisms in

Milk: The Breed Count

Companies, 2001

67

Direct Microscopic Count of Organisms

in Milk:

The Breed Count

has five one-centimeter areas that are surrounded byground glass, obviating the need for a card Proceed asfollows:

Materials:

Breed slide or guide cardBreed pipettes (0.01 ml)methylene blue, xylol, 95% alcoholbeaker of water and electric hot platesamples of raw milk (poor and high quality)

1 Shake the milk sample 25 times to completely perse the organisms and break up large clumps ofbacteria

dis-2 Transfer 0.01 ml of milk to one square on theslide The pipette may be filled by capillary ac-tion or by suction, depending on the type ofpipette The instructor will indicate which

method to use Be sure to wipe off the outside tip

of the pipette with tissue before touching the slide

to avoid getting more than 0.01 ml on the slide

3 Allow the slide to air-dry and then place it over

a beaker of boiling water for 5 minutes to fix it

steam-4 Flood the slide with xylol to remove fat globules.

5 Remove the xylol from the slide by flooding the

slide with 95% ethyl alcohol.

6 Gently immerse the slide into a beaker of distilled water to remove the alcohol Do not hold it under

running water; the milk film will wash off

When it is necessary to determine milk quality in a

much shorter time than is possible with a standard

plate count, one can make a direct microscopic

count on a slide This is accomplished by staining a

measured amount of milk that has been spread over an

area one square centimeter on a slide The slide is

ex-amined under oil and all of the organisms in an entire

microscopic field are counted To increase accuracy,

several fields are counted to get average field counts

Before the field counts can be translated into

organ-isms per milliliter, however, it is necessary to

calcu-late the field area

High-quality milk will have very few organisms

per field, necessitating the examination of many

fields A slide made of poor-quality milk, on the other

hand, will reveal large numbers of bacteria per field,

thus requiring the examination of fewer fields An

ex-perienced technician can determine, usually within

15 minutes, whether or not the milk is of acceptable

quality

In addition to being much faster than the SPC, the

direct microscopic count has two other distinct

ad-vantages First of all, it will reveal the presence of

bacteria that do not form colonies on an agar plate at

35° C; thermophiles, psychrophiles, and dead bacteria

would fall in this category Secondly, the presence of

excessive numbers of leukocytes and pus-forming

streptococci on a slide will be evidence that the

ani-mal that produced the milk has an udder infection

(mastitis)

In view of all these advantages, it is apparent that

the direct microscopic count has real value in milk

testing It is widely used for testing raw milk in

creamery receiving stations and for diagnosing the

types of contamination and growth in pasteurized

milk products

In this exercise, samples of raw whole milk will

be examined Milk that has been separated, blended,

homogenized, and pasteurized will lack leukocytes

and normal flora

There are several acceptable ways of spreading the

milk onto the slide Figure 67.1 illustrates a method

using a guide card The Breed slide used in figure 67.2

Figure 67.1 Using a guide card to spread milk sample over one square centimeter on a slide

Applications Lab Manual,

Eighth Edition

and Food Products Count of Organisms in

Milk: The Breed Count

Companies, 2001

7 Stain the smear with methylene blue for 15

sec-onds and dip the slide again in water to remove

the excess stain

8 Decolorize the smear to pale blue with 95%

alco-hol and dip in distilled water to stop decolorization

9 Allow the slide to completely air-dry before

ex-amination

(Microscope Factor [MF])

Before counting the organisms in each field it is

nec-essary to know what part of a milliliter of milk is

rep-resented in that field The relationship of the field to a

milliliter is the microscope factor (MF) To calculate

the MF, it is necessary to use a stage micrometer to

measure the diameter of the oil immersion field By

applying the formula ␲r2

to this measurement, thearea is easily determined With the amount of milk

(0.01 ml) and the area of the slide (1 cm2), it is a

sim-ple matter to calculate the MF

Materials:

stage micrometer

1 Place a stage micrometer on the microscope stageand bring it into focus under oil Measure the di-ameter of the field, keeping in mind that eachspace is equivalent to 0.01 mm

2 Calculate the area of the field in square ters, using the formula ␲r2(␲ ⫽ 3.14)

millime-3 Convert the area of the field from square limeters to square centimeters by dividing by 100

mil-4 Calculate the number of fields in one square timeter by dividing one square centimeter by thearea of the field in square centimeters

cen-5 To get the part of a milliliter that is represented in

a single field (microscope factor), multiply the

number of fields by 100 The value should bearound 500,000 Therefore, a single field repre-sents 1/500,000 of a ml of milk Record yourcomputations on the Laboratory Report

Two methods of counting the bacteria can be used: dividual cells may be tallied or only clumps of bacte-ria may be counted In both cases, the number per mil-liliter will be higher than a standard plate count, but a

in-Exercise 67 • Direct Microscopic Count of Organisms in Milk: The Breed Count

Applications Lab Manual,

Eighth Edition

and Food Products Count of Organisms in

Milk: The Breed Count

Companies, 2001

clump count will be closer to the SPC Both methods

will be used

1 After the microscope has been calibrated, replace

the stage micrometer with the stained slide

Examine it under oil immersion optics

2 Count the individual cells in five fields and record

your results on the Laboratory Report A field is the

entire area encompassed by the oil immersion lens

As you see leukocytes, record their numbers, also

3 Count only clumps of bacteria in five fields,recording the numbers of leukocytes as well.Record the totals on the Laboratory Report

4 Calculate the number of organisms, clumps, andbody cells per milliliter using the microscope factor

Complete the last portion of Laboratory Report 66, 67

Direct Microscopic Count of Organisms in Milk: The Breed Count • Exercise 67

Clean high-grade milk will have very few, if any, bacteria.

Milk from a cow with mastitis Long chain streptococci

and numerous leukocytes are visible.

High-grade milk that is allowed to stand without cooling will reveal numerous streptococci as short chains and diplococci.

Milk that is placed in improperly cleaned utensils will exhibit masses of miscellaneous bacteria.

Figure 67.3 Microscopic fields of milk samples

Applications Lab Manual,

Eighth Edition

Reductase Test

68

Milk that contains large numbers of actively growing

bacteria will have a lowered oxidation-reduction

po-tential due to the exhaustion of dissolved oxygen by

microorganisms The fact that methylene blue loses

its color (becomes reduced) in such an environment

is the basis for the reductase test In this test, 1 ml of

methylene blue (1:25,000) is added to 10 ml of milk

The tube is sealed with a rubber stopper and slowly

inverted three times to mix It is placed in a water

bath at 35° C and examined at intervals up to 6 hours

The time it takes for the methylene blue to become

colorless is the methylene blue reduction time

(MBRT) The shorter the MBRT, the lower the

qual-ity of milk An MBRT of 6 hours is very good Milkwith an MBRT of 30 minutes is of very poor quality.The validity of this test is based on the assump-tion that all bacteria in milk lower the oxidation-reduction potential at 35° C Large numbers of psy-chrophiles, thermophiles, and thermodurics, which donot grow at this temperature, would not produce apositive test Raw milk, however, will contain pri-

marily Streptococcus lactis and Escherichia coli,

which are strong reducers; thus, this test is suitable forscreening raw milk at receiving stations Its principalvalue is that less technical training of personnel is re-quired for its performance

Methylene Blue

Rubber Stopper

GOOD QUALITY MILK Methylene blue is not reduced within 6 hours.

35° C Water Bath

POOR QUALITY MILK Methylene blue is reduced within 2 hours.

Applications Lab Manual,

Eighth Edition

In this exercise, samples of low- and high-quality

raw milk will be tested

1 Attach gummed labels with your name and type

of milk to two test tubes Each student will test a

good-quality as well as a poor-quality milk

2 Using separate 10 ml pipettes for each type of

milk, transfer 10 ml to each test tube To the milk

in the tubes add 1 ml of methylene blue with a 1

ml pipette Insert rubber stoppers and gently

in-vert three times to mix Record your name and thetime on the labels and place the tubes in the waterbath, which is set at 35° C

3 After 5 minutes incubation, remove the tubesfrom the bath and invert once to mix This is thelast time they should be mixed

4 Carefully remove the tubes from the water bath

30 minutes later and every half hour until the end

of the laboratory period When at least four-fifths

of the tube has turned white, the end point of

re-duction has taken place Record this time on theLaboratory Report The classification of milkquality is as follows:

Class 1: Excellent, not decolorized in 8 hours Class 2: Good, decolorized in less than 8 hours,

but not less than 6 hours

Class 3: Fair, decolorized in less than 6 hours,

but not less than 2 hours

Class 4: Poor, decolorized in less than 2 hours.

The Reductase Test • Exercise 68

Applications Lab Manual,

Eighth Edition

Bacterial Counts of Foods

69

The standard plate count, as well as the multiple tube

test, can be used on foods much in the same manner that

they are used on milk and water to determine total counts

and the presence of coliforms To get the organisms in

suspension, however, a food blender is necessary

In this exercise, samples of ground meat, dried

fruit, and frozen food will be tested for total numbers

of bacteria This will not be a coliform count The

in-structor will indicate the specific kinds of foods to be

tested and make individual assignments Figure 69.1

illustrates the general procedure

Materials:

per student:

3 Petri plates

1 bottle (45 ml) of Plate Count agar or

Standard Methods agar

1 99 ml sterile water blank

2 1.1 ml dilution pipettes

per class:

food blender

sterile blender jars (one for each type of food)

sterile weighing paper

180 ml sterile water blanks (one for each type

of food)samples of ground meat, dried fruit, and frozenvegetables, thawed 2 hours

1 Using aseptic techniques, weigh out on sterileweighing paper 20 grams of food to be tested

2 Add the food and 180 ml of sterile water to a ile mechanical blender jar Blend the mixture for

ster-5 minutes This suspension will provide a 1:10dilution

3 With a 1.1 ml dilution pipette dispense from theblender 0.1 ml to plate I and 1.0 ml to the waterblank See figure 69.1

4 Shake the water blank 25 times in an arc for 7 onds with your elbow on the table as done inExercise 23 (Bacterial Population Counts)

sec-5 Using a fresh pipette, dispense 0.1 ml to plate IIIand 1.0 ml to plate II

6 Pour agar (50° C) into the three plates and bate them at 35° C for 24 hours

incu-7 Count the colonies on the best plate and recordthe results on the Laboratory Report

Applications Lab Manual,

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70

Microbial Spoilage of Canned Food

tive containers that permit the entrance of organismsafter the heat process

Our concern here will be with the most commontypes of food spoilage caused by heat-resistant spore-forming bacteria There are three types: “flat sour,”

“T.A spoilage,” and “stinker spoilage.”

Flat sour pertains to spoilage in which acids are

formed with no gas production; result: sour food in

cans that have flat ends T.A spoilage is caused by

thermophilic anaerobes that produce acid and gases(CO2and H2, but not H2S) in low-acid foods Cans

swell to various degrees, sometimes bursting Stinker spoilage is due to spore-formers that produce hydro-

gen sulfide and blackening of the can and contents.Blackening is due to the reaction of H2S with the iron

in the can to form iron sulfide

In this experiment you will have an opportunity tobecome familiar with some of the morphological and

Spoilage of heat-processed, commercially canned

foods is confined almost entirely to the action of

bac-teria that produce heat-resistant endospores Canning

of foods normally involves heat exposure for long

pe-riods of time at temperatures that are adequate to kill

spores of most bacteria Particular concern is given to

the processing of low-acid foods in which

Clostridium botulinum can thrive to produce botulism

food poisoning

Spoilage occurs when the heat processing fails to

meet accepted standards This can occur for several

reasons: (1) lack of knowledge on the part of the

processor (usually the case in home canning); (2)

carelessness in handling the raw materials before

can-ning, resulting in an unacceptably high level of

con-tamination that ordinary heat processing may be

inad-equate to control; (3) equipment malfunction that

results in undetected underprocessing; and (4)

defec-Each can of corn or peas is

perforated with an awl or ice pick.

To create an air space under the cover, some liquid is poured off.

Contents of each can is inoculated with one of five different organisms.

Hole in each can is sealed by soldering over it.

24–48 Hours Incubation

For Temperature See text

1 Type of spoilage caused by each

orga-nism is noted.

2 Gram- and spore-stained slides are made

from contents of cans.

SECOND PERIOD

Figure 70.1 Canned food inoculation procedure

Applications Lab Manual,

Eighth Edition

physiological characteristics of organisms that cause

canned food spoilage, including both aerobic and

anaer-obic endospore formers of Bacillus and Clostridium, as

well as a non-spore-forming bacterium

Working as a single group, the entire class will

in-oculate 10 cans of vegetables (corn and peas) with

five different organisms Figure 70.1 illustrates the

procedure Note that the cans will be sealed with

sol-der after inoculation and incubated at different

tem-peratures After incubation the cans will be opened so

that stained microscope slides can be made to

deter-mine Gram reaction and presence of endospores Your

instructor will assign individual students or groups of

students to inoculate one or more of the 10 cans One

can of corn and one can of peas will be inoculated

with each of the organisms Proceed as follows:

(Inoculations)

Materials:

5 small cans of corn

5 small cans of peas

cultures of B stearothermophilus,

B coagulans, C sporogenes,

C thermosaccharolyticum, and E coli

ice picks or awls

hammer

solder and soldering iron

plastic bags

gummed labels and rubber bands

1 Label the can or cans with the name of the

organ-ism that has been assigned to you Use white

gummed labels In addition, place a similar label

on one of the plastic bags to be used after sealing

of the cans

2 With an ice pick or awl, punch a small hole

through a flat area in the top of each can This can

be done easily with the heel of your hand or a

hammer, if available

3 Pour off a small amount of the liquid from the can

to leave an air space under the lid

4 Use an inoculating needle to inoculate each can of

corn or peas with the organism indicated on the

label

5 Take the cans up to the demonstration table where

the instructor will seal the hole with solder

6 After sealing, place each can in two plastic bags.Each bag must be closed separately with rubberbands, and the outer bag must have a label on it

7 Incubation will be as follows till the next period:

• 55° C—C thermosaccharolyticum and

B stearothermophilus

• 37° C—C sporogenes and B coagulans

• 30° C — E coli

Note: If cans begin to swell during incubation,

they should be placed in refrigerator

(Interpretation)

After incubation, place the cans under a hood to openthem The odors of some of the cans will be verystrong due to H2S production

Materials:

can opener, punch typesmall plastic beakersParafilm

gram-staining kitspore-staining kit

1 Open each can carefully with a punch-type canopener If the can is swollen, hold an invertedplastic funnel over the can during perforation tominimize the effects of any explosive release ofcontents

2 Remove about 10 ml of the liquid through theopening, pouring it into a small plastic beaker.Cover with Parafilm This fluid will be used formaking stained slides

3 Return the cans of food to the plastic bags, reclosethem, and dispose in a proper trash bin

4 Prepare gram-stained and endospore-stainedslides from your canned food extract as well asfrom the extracts of all the other cans Examineunder brightfield oil immersion

5 Record your observations on the report sheet onthe demonstration table It will be duplicated and

a copy will be made available to each student

Complete the first portion of Laboratory Report 70, 71

Exercise 70 • Microbial Spoilage of Canned Food

Applications Lab Manual,

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and Food Products Refrigerated Meats Companies, 2001

71

Microbial Spoilage of Refrigerated Meat

Aeromonas hydrophila, Clostridium botulinum, teria monocytogenes, Vibrio cholera, Yersinia enter- colitica, and some strains of E coli.

Lis-In addition to bacterial spoilage of meat there aremany yeasts and molds that are psychrophilic and psy-chrotrophic Examples of psychrophilic yeasts are

Cryptococcus, Leucosporidium, and Torulopsis.

Psychrotrophic fungi include Candida, Cryptococcus,

Saccharomyces, Alternaria, Aspergillus, Cladosporium, Fusarium, Mucor, Penicillium, and many more.

Our concern in this experiment will be to testone or more meat samples for the prevalence of psychrophilic-psychrotrophic organisms To accom-plish this, we will liquefy and dilute out a sample ofground meat so that it can be plated out and then in-cubated in a refrigerator for 2 weeks After incuba-tion, colony counts will be made to determine thenumber of organisms of this type that exist in a gram

sterile Petri dish or sterile filter paper

per pair of students:

4 large test tubes of sterile phosphate bufferedwater (9 ml each)

4 TSA plates

9 sterile 1 ml pipettesL-shaped glass spreading rodbeaker of 95% ethyl alcohol

At Demonstration Table

1 With a sterile scoopula, weigh 10g of ground meatinto a sterile Petri plate or onto a sterile piece offilter paper

Contamination of meats by microbes occurs during

and after slaughter Many contaminants come from

the animal itself, others from utensils and

equip-ment The conditions for rapid microbial growth in

freshly cut meats are very favorable, and spoilage

can be expected to occur rather quickly unless steps

are taken to prevent it Although immediate

refriger-ation is essential after slaughter, it will not prevent

spoilage indefinitely, or even for a long period of

time under certain conditions In time, cold-tolerant

microbes will destroy the meat, even at low

refriger-ator temperatures

Microorganisms that grow at temperatures

be-tween 5° and 0° C are classified as being either

psy-chrophilic or psychrotrophic The difference

be-tween the two groups is that psychrophiles seldom

grow at temperatures above 22° C and

psy-chrotrophs (psychrotolerants or low-temperature

mesophiles) grow well above 25° C While the

opti-mum growth temperature range for psychrophiles is

15°–18° C, psychrotrophs have an optimum growth

temperature range of 25°–30° C It is the

psy-chrotrophic microorganisms that cause most meat

spoilage during refrigeration

The majority of psychrophiles are gram-negative

and include species of Aeromonas, Alcaligenes,

Cytophagia, Flavobacterium, Pseudomonas, Serratia,

and Vibrio Gram-positive psychrophiles include

species of Arthrobacter, Bacillus, Clostridium, and

Micrococcus.

Psychrotrophs include a much broader spectrum

of gram-positive and gram-negative rods, cocci,

vibrios, spore-formers, and non-spore-formers

Typi-cal genera are Acinetobacter, Chromobacterium,

Cit-robacter, Corynebacterium, EnteCit-robacter,

Escheri-chia, Klebsiella, Lactobacillus, Moraxella,

Staphy-lococcus, and Streptococcus.

The widespread use of vacuum or modified

at-mospheric packaging of raw and processed meat has

resulted in food spoilage due to facultative and

obli-gate anaerobes, such as Lactobacillus, Leuconostoc,

Pediococcus, and certain Enterobacteriaceae.

Although most of the previously mentioned

psy-chrotrophic representatives are nonpathogens, there

are significant pathogenic psychrotrophs such as

Applications Lab Manual,

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and Food Products Refrigerated Meats Companies, 2001

2 Pour 90 ml of sterile buffered water from water

blank into a sterile blender jar and add the meat

3 Blend the meat and water at moderate speed for 1

minute.

Student Pair

1 Label the four water blanks 1 through 4

2 Label the four Petri plates with their dilutions, as

indicated in figure 71.1 Add your initials and

date also

3 Once blender suspension is ready, pipette 1 ml

from jar to tube 1

4 Using a fresh 1 ml pipette, mix the contents in

tube 1 and transfer 1 ml to tube 2

5 Repeat step 4 for tubes 3 and 4, using fresh

pipettes for each tube.

6 Dispense 0.1 ml from each tube to their respective

plates of TSA Note that by using only 0.1 ml per

plate you are increasing the dilution factor by 10

times in each plate

7 Using a sterile L-shaped glass rod, spread the

or-ganisms on the agar surfaces Sterilize the rod

each time by dipping in alcohol and flaming tly Be sure to let rod cool completely each time

gen-8 Incubate the plates for 2 weeks in the back of therefrigerator (away from door-opening) where thetemperature will remain between 0° and 5° C

Materials:

Quebec colony countershand tally countersgram-staining kit

1 After incubation, count the colonies on all theplates and calculate the number of psychrophilesand psychrotrophs per gram of meat

2 Select a colony from one of the plates and prepare

a gram-stained slide Examine under oil sion and record your observations on theLaboratory Report

Complete the last portion of Laboratory Report 70, 71

Exercise 71 • Microbial Spoilage of Refrigerated Meat

A tenfold serial dilution is made by transferring 1 ml from each tube to the next one.

1 ml Ten grams of ground meat is

added to 90 ml of water and

blended for 1 minute.

1:10

An alcohol-flamed glass rod is used to spread

After spreading out of organisms on the agar surfaces, the plates are incubated at 0 °–5° C for

Applications Lab Manual,

or white grapes can be used, but the skins are carded White and red wines are fermented at 13° C(55° F) and 24° C (75° F), respectively

dis-In this exercise we will set up a grape juice mentation experiment to learn about some of the char-acteristics of sugar fermentation to alcohol Note infigure 72.1 that a balloon will be attached over themouth of the fermentation flask to exclude oxygen up-take and to trap gases that might be produced To de-tect the presence of hydrogen sulfide production wewill tape a lead acetate test strip inside the neck of theflask The pH of the substrate will also be monitoredbefore and after the reaction to note any changes thatoccur

fer-Fermented food and beverages are as old as

civiliza-tion Historical evidence indicates that beer and wine

making were well established as long ago as 2000 B.C.

An Assyrian tablet states that Noah took beer aboard

the ark

Beer, wine, vinegar, buttermilk, cottage cheese,

sauerkraut, pickles, and yogurt are some of the more

commonly known products of fermentation Most of

these foods and beverages are produced by different

strains of yeasts (Saccharomyces) or bacteria

(Lactobacillus, Acetobacter, etc.).

Fermentation is actually a means of food

preser-vation because the acids formed and the reduced

en-vironment (anaerobiasis) hold back the growth of

many spoilage microbes

Wine is essentially fermented fruit juice in which

alcoholic fermentation is carried out by Saccharomyces

cerevisiae var ellipsoideus Although we usually

asso-ciate wine with fermented grape juice, it may also be

made from various berries, dandelions, rhubarb, etc

Three conditions are necessary: simple sugar, yeast,

and anaerobic conditions The reaction is as follows:

Figure 72.1 Alcohol fermentation setup

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and Food Products Alcohol Fermentation Companies, 2001

Materials:

100 ml grape juice (no preservative)

bottle of juice culture of wine yeast

3 Determine the pH of the juice with a pH meter

and record the pH on the Laboratory Report

4 Agitate the container of yeast juice culture to

sus-pend the culture, remove 5 ml with a pipette, and

add it to the flask

5 Attach a short strip of tape to a piece of lead-acetate

test paper (3 cm long), and attach it to the inside

surface of the neck of the flask Make certain that

neither the tape nor the test strip protrudes from the

flask

6 Cover the flask opening with a balloon

7 Incubate at 15°–17° C for 2–5 days

2 Determine the pH and record it on the LaboratoryReport

3 Record any change in color of the lead-acetate-teststrip on the Laboratory Report If any H2S is pro-duced, the paper will darken due to the formation

of lead sulfide as hydrogen sulfide reacts with thelead acetate

4 Wash out the flask and return it to the drain rack

Complete the first portion of Laboratory Report 72,

73 by answering all the questions

Exercise 72 • Microbiology of Alchohol Fermentation

Applications Lab Manual,

Eighth Edition

73

Microbiology of Yogurt Production

In this exercise you will produce a batch of yogurtfrom milk by using an inoculum from commercial yo-gurt Gram-stained slides will be made from the fin-ished product to determine the types of organisms thatcontrol the reaction If proper safety measures are fol-lowed, the sample can be tasted

Two slightly different ways of performing this periment are provided here Your instructor will indi-cate which method will be followed

(First Period)

Figure 73.1 illustrates the procedure for this method.Note that 4 g of powdered milk are added to 100 ml ofwhole milk This mixture is then heated to boiling andcooled to 45°C After cooling, the milk is inoculated withyogurt and incubated at 45° C for 24 hours Proceed:

For centuries, people throughout the world have

been producing fermented milk products using

yeasts and lactic acid–producing bacteria The

yo-gurt of eastern central Europe, the kefir of the

Cossacks, the koumiss of central Asia, and the leben

of Egypt are just a few examples In all of these

fer-mented milks, lactobacilli act together with some

other microorganisms to curdle and thicken milk,

producing a distinctive flavor desired by the

pro-ducer Kefir of the Cossacks is made by charging

milk with small cauliflower-like grains that contain

Streptococcus lactis, Saccharomyces delbrueckii,

and Lactobacillus brevis As the grains swell in the

milk they release the growing microorganisms to

fer-ment the milk The usual method for producing

yo-gurt in large-scale production is to add pure cultures

of Streptococcus thermophilus and Lactobacillus

bulgaricus to pasteurized milk.

Dried Milk Powder

Four grams of dried milk powder is dissolved in 100 ml of whole milk.

2.

1 Product is evaluated with respect

to texture, color, aroma, and taste.

Slides, stained with methylene blue, are studied to determine morphology of organisms.

Milk is brought to boiling point while stirring constantly.

3

Figure 73.1 Yogurt production by Method A

Applications Lab Manual,

commercial yogurt (with viable organisms)

small beaker, graduate, teaspoon, stirring rod

plastic wrap

filter paper (for weighing)

1 On a piece of filter paper weigh 4 grams of dried

powdered milk

2 To a beaker of 100 ml of whole milk add the

pow-dered milk and stir thoroughly with sterile glass

rod to dissolve

3 Heat to boiling, while stirring constantly

4 Cool to 45° C and inoculate with 1 teaspoon of the

commercial yogurt Stir Be sure to check the

la-bel to make certain that product contains a live

culture Cover with plastic wrap

5 Incubate at 45° C for 24 hours

(First Period)

Figure 73.2 illustrates a slightly different method of

culturing yogurt, which, due to its simplicity, may be

preferred Note that no whole milk is used and

provi-sions are made for producing a sample for tasting

Materials:

small beaker, graduate, teaspoon, stirring rod

dried powdered milk

commercial yogurt (with viable organisms)

plastic wrap

filter paper for weighing

paper Dixie cup (5 oz size) and coverelectric hot plate or Bunsen burner and tripod

1 On a piece of filter paper weigh 25 grams of driedpowdered milk

2 Heat 100 ml of water in a beaker to boiling andcool to 45° C

3 Add the 25 grams of powdered milk and 1 spoon of yogurt to the beaker of water Mix the in-gredients with a sterile glass rod

tea-4 Pour some of the mixture into a sterile Dixie cupand cover loosely Cover the remainder in thebeaker with plastic wrap

5 Incubate at 45° C for 24 hours

(Both Methods)

1 Examine the product and record on the LaboratoryReport the color, aroma, texture, and, if desired,the taste

2 Make slide preparations of the yogurt culture Fixand stain with methylene blue Examine under oilimmersion and record your results on LaboratoryReport 72, 73

Complete the last portion of Laboratory Report 72, 73

by answering all the questions

Exercise 73 • Microbiology of Yogurt Production

a clean small beaker.

Water is cooled down

to 45° C.

2 Twenty-five grams of dried powdered milkand a teaspoonful of commercial yogurt are stirred into the 100 ml of water at 45° C.

Applications Lab Manual,

Eighth Edition

Bacterial Genetic Variations

Variations in bacteria that are due to environmental factors and that

do not involve restructuring DNA are designated as temporary variations Such variations may be morphological or physiological

and disappear as soon as the environmental changes that brought

them about disappear For example, as a culture of E coli becomes

old and the nutrients within the tube become depleted, the newcells that form become so short that they appear coccoidal.Reinoculation of the organism into fresh media, however, results inthe reappearance of distinct bacilli of characteristic length.Variations in bacteria that involve alteration of the DNA macro-

molecule are designated as permanent variations It is because

they survive a large number of transfers that they are so named.Such variations are due to mutations Variations of this type occurspontaneously They also might be induced by physical and chem-ical methods Some permanent variations also are caused by thetransfer of DNA from one organism to another, either directly byconjugation or indirectly by phage It is these permanent geneticvariations that the three exercises of this unit represent

Exercises 74 and 75 of this unit demonstrate how spontaneousmutations are constantly occurring in bacterial populations Thegenetic change that occurs in these two exercises pertains to thedevelopment of bacterial resistance to streptomycin In Exercise 76

we will study how chemically induced mutagenicity that causesback mutations is used in the Ames test to determine possible car-cinogenicity of chemical compounds

13

Applications Lab Manual,

Eighth Edition

Mutant Isolation by Gradient Plate Method

74

An excellent way to determine the ability of

organ-isms to produce mutants that are resistant to

antibi-otics is to grow them on a gradient plate of a

par-ticular antibiotic Such a plate consists of two

different wedgelike layers of media: a bottom layer

of plain nutrient agar and a top layer of nutrient agar

with the antibiotic Since the antibiotic is only in the

top layer, it tends to diffuse into the lower layer,

pro-ducing a gradient of antibiotic concentration from

low to high

In this exercise we will make a gradient plate

us-ing streptomycin in the medium E coli, which is

nor-mally sensitive to this antibiotic, will be spread over

the surface of the plate and incubated for 4 to 7 days

Any colonies that develop in the high concentration

area will be streptomycin-resistant mutants

The gradient plate used in this experiment will have a

high concentration of 100 mcg of streptomycin per

milliliter of medium This concentration is 10 times

the strength used in sensitivity disks in the

Kirby-Bauer test method Prepare a gradient plate as follows:

Materials:

1 sterile Petri plate

2 nutrient agar pours (10 ml per tube)

1 tube of streptomycin solution (1%)

3 Remove the wood spacer from under the plate

4 Pipette 0.1 ml of streptomycin into second agarpour, mix tube between palms, and pour contentsover medium of plate that is now resting level onthe table

5 Label the low and high concentration areas on thebottom of the plate

The inoculation procedure is illustrated in figure 74.2.The technique involves spreading a measured amount

Applications Lab Manual,

Eighth Edition

of the culture on the surface of the medium with a

glass bent rod to provide optimum distribution

Materials:

1 beaker of 95% ethanol

1 glass rod spreader

nutrient broth culture of E coli

1 ml pipette

1 Pipette 0.1 ml of E coli suspension onto surface

of medium in Petri plate

2 Sterilize glass spreading rod by dipping it in

alco-hol first and then passing it quickly through the

flame of a Bunsen burner Cool the rod by placing

against sterile medium in plate before contacting

organisms

3 Spread the culture evenly over the surface with

the glass rod

4 Invert and incubate the plate at 37° C for 4 to 7

days in a closed cannister or plastic bag Unless

incubated in this manner, excessive dehydration

might occur

After 4 to 7 days, look for colonies of E coli in the

area of high streptomycin concentration Count thecolonies that appear to be resistant mutants and recordyour count on the Laboratory Report

Select a well-isolated colony in the high tration area and, with a sterile loop, smear the colonyover the surface of the medium toward the higher con-centration portion of the plate Do this with two orthree colonies Return the plate to the incubator foranother 2 or 3 days

Examine the plate again to note what effect thespreading of the colonies had on their growth Recordyour observations on Laboratory Report 74, 75

Mutant Isolation by Gradient Plate Method • Exercise 74

Spreading rod is dipped in ethanol for cleaning.

Organisms are spread evenly over surface of agar.

Rod is sterilized in Bunsen burner flame.

Figure 74.2 Procedure for spreading organisms on gradient plate

Applications Lab Manual,

Eighth Edition

Mutant Isolation by Replica Plating

75

In the last exercise it was observed that E coli could

develop mutant strains that are streptomycin-resistant

If we had performed this experiment with other

organ-isms and with other antibiotics, the results would have

been quite similar The question that logically

devel-ops in one’s mind from this experiment is: What

mech-anism is involved here? Is a mutation of this sort

in-duced by the antibiotic? Or does the mutation occur

spontaneously and independently of the presence of

the drug? If we could demonstrate the presence of a

streptomycin-resistant mutant occurring on a medium

that lacks streptomycin, then we could assume that the

mutation occurs spontaneously

To determine whether or not such a colony exists

on a plain agar plate having 500 to 1,000 colonies

could be a laborious task One would have to transfer

organisms from each colony to a medium containingstreptomycin This is somewhat self-defeating, too, inlight of the low incidence of mutations that occur.Many thousands of the transfers might have to bemade to find the first mutant Fortunately, we can re-sort to replica plating to make all the transfers in onestep Figure 75.1 illustrates the procedure In thistechnique a velveteen-covered colony transfer device

is used to make the transfers

Note in figure 75.1 that organisms are first persed on nutrient agar with a glass spreading rod.After incubation, all colonies are transferred from thenutrient agar plate to two other plates: first to a nutri-ent agar plate and second to a streptomycin agar plate.After incubation, streptomycin-resistant strains arelooked for on the streptomycin agar

dis-Organisms are spread over nutrient agar with a steril bent glass rod.

After incubation, colonies are picked

up with velveteen colony carrier.

Applications Lab Manual,

Eighth Edition

Materials:

1 Petri plate of nutrient agar

1 bent glass spreading rod

1 ml serological pipette

beaker of 95% ethanol

Bunsen burner

broth culture of E coli

1 Pipette 0.1 ml of E coli from broth culture to

sur-face of medium in Petri dish

2 With a sterile bent glass rod, spread the organisms

over the plate following the routine shown in

1 Petri plate of nutrient agar per student

1 Petri plate of streptomycin agar (100 micrograms of streptomycin per ml

of medium)

1 sterile colony carrier per student

1 Carefully lower the sterile colony carrier onto the

colonies of E coli on the plate from the previous

period

2 Inoculate the plate of nutrient agar by lightlypressing the carrier onto the medium

3 Now without returning the carrier to the original

culture plate, inoculate the streptomycin agar inthe same manner

4 Incubate both plates at 37° C for 2 to 4 days in anenclosed cannister

Materials:

Quebec colony counter and hand counter

1 Examine both plates and record the informationcalled for on Laboratory Report 74, 75

2 Tabulate the results of other members of the class

Mutant Isolation by Replica Plating • Exercise 75

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Variations and Carcinogenesis: The

Ames Test

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Bacterial Mutagenicity and Carcinogenesis:

The Ames Test

76

The fact that carcinogenic compounds induce

in-creased rates of mutation in bacteria has led to the use

of bacteria for screening chemical compounds for

possible carcinogenesis The Ames test, developed

by Bruce Ames at the University of California–

Berkeley, has been widely used for this purpose

The conventional way to determine whether a

chemical substance is carcinogenic is to inject the

material into animals and look for the development of

tumors If tumors develop, it is presumed that the

substance can cause cancer Although this method

works well, it is costly, time-consuming, and

cum-bersome, especially if it is applied to all the industrial

chemicals that have found their way into food and

water supplies

The Ames test serves as a screening test for the

detection of carcinogenic compounds by testing the

ability of chemical agents to induce bacterial

muta-tions Although most mutagenic agents are

carcino-genic, some are not; however, the correlation between

carcinogenesis and mutagenicity is high—around

83% Once it has been determined that a specific

agent is mutagenic, it can be used in animal tests to

confirm its carcinogenic capability

The standard way to test chemicals for

mutagene-sis has been to measure the rate of back mutations in

strains of auxotrophic bacteria In the Ames test a

strain of Salmonella typhimurium, which is

aux-otrophic for histidine (unable to grow in the absence of

histidine), is exposed to a chemical agent After

chem-ical exposure and incubation on histidine-deficient

medium, the rate of reversion (back mutation) to

pro-totrophy is determined by counting the number of

colonies that are seen on the histidine-deficient

medium

Although testing of chemicals for mutagenesis in

bacteria has been performed for a long time, two new

features are included in the Ames test that make it a

powerful tool The first is that the strain of S

ty-phimurium used here lacks DNA repair enzymes,

which prevents the correction of DNA injury The

sec-ond feature of the test is the incorporation of

mam-There are two ways to perform the Ames test The

method illustrated in figure 76.1 is a spot test that is

widely used for screening purposes The other method

is the plate incorporation test, which is used for

quantitative analysis of the mutagenic effectiveness

of compounds Our concern here will be with the spottest; however, since the concentration of the liver ex-tract is very critical, we will omit using it in our test.The test, as performed here, will work well without it.Success in performing the spot test requires con-siderable attention to careful measurements and tim-ing It is for this reason that students will work in pairs

to perform the test

Note in figure 76.1 that 0.1 ml of S typhimurium

is first added to a small tube that contains 2 ml of topagar that is held at 45° C This top agar contains

0.6% agar, 0.5% NaCl, and a trace of histidine and

biotin The histidine allows the bacteria to gothrough several rounds of cell division, which is es-sential for mutagenesis to occur Since the histidinedeletion extends through the biotin gene, biotin isalso needed This early growth of cells produces afaint background lawn that is barely visible to thenaked eye

Before pouring the top agar over the glucose–minimal salts agar, the tube must be vortexed at slowspeed for 3 seconds and poured quickly to get evendistribution The addition of the bacteria, vortexing,and pouring must be accomplished in 20 seconds.Failure to move quickly enough will cause stippling

of the top agar

There are two ways that one can use to apply thechemical agent to the top agar: a filter paper disk may

be used, or the chemical can be applied directly to thecenter of the plate without a disk The procedureshown in figure 76.1 involves using a disk

Note the unusual way in which a filter paper disk

is impregnated in figure 76.1 To get it to stand onedge it must be put in position with sterile forceps andpressed in slightly to hold it upright Just the rightamount of the chemical agent is then added with aPasteur pipette to the upper edge of the disk to com-

Applications Lab Manual,

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Variations and Carcinogenesis: The

Ames Test

Companies, 2001

Bacterial Mutagenicity and Carcinogenesis: The Ames Test • Exercise 76

Figure 76.1 Procedure for performing a modified Ames test

Applications Lab Manual,

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Variations and Carcinogenesis: The

Ames Test

Companies, 2001

agent is mutagenic, a halo of densely packed revertant

colonies will be seen around the disk Scattered larger

colonies will show up beyond the halo that represent

spontaneous back mutations, not related to the test

reagent

You will be issued an unknown chemical agent to

test and you will have an opportunity to test some

other substance you have brought to the laboratory In

addition to these two tests you will be inoculating

pos-itive and negative test controls: thus, each pair of

stu-dents will be responsible for four plates

Keep in mind as you perform this experiment that

there is a lot more to the Ames test than revealed here

While we are using only one tester strain of S

ty-phimurium, there are several others that are used in

routine testing The additional strains are needed to

accommodate different kinds of chemical

com-pounds While one chemical agent may be mutagenic

on one tester strain, it may produce a negative result

on another strain Also, keep in mind that we are not

taking advantage of using the liver extract

(Inoculations)

Materials:

per pair of students:

4 plates of glucose–minimal salts agar

(30 ml per plate)

4 tubes of top agar (2 ml per tube)

tube of sterile water

Vortex mixer

sterile Pasteur pipettes, forceps

serological pipettes (1 ml size)

filter paper disks, sterile in Petri dish

test reagents:

4-NOPD (10 ␮g/ml) solution*

tube of unknown possible carcinogen

substance from home for testing

culture of S typhimurium, Ames strain, TA98 in

trypticase soy broth

*4-nitro-o-phenylenediamine

1 Working with your laboratory partner, label the

bottoms of four glucose–minimal salts agar plates

as follows: POSITIVE CONTROL, NEGATIVE

CONTROL, UNKNOWN, and OPTIONAL

2 Liquefy four tubes of top agar and cool to 45° C

3 With a 1 ml serological pipette, inoculate a tube

of top agar with 0.1 ml of S typhimurium.

rolling the tube between the palms of both hands.Pour the contents onto the positive control plate

of glucose–minimal salts agar The agar plate

must be at room temperature Work rapidly to achieve pipetting, mixing, and spreading in 20 seconds.

5 Repeat steps 3 and 4 for each of the other threetubes of top agar

6 With sterile forceps place a disk on its edge near

the center of the positive control plate Sterilize

the forceps by dipping in alcohol and flaming

7 With a sterile Pasteur pipette, deposit just enough4-NOPD on the upper edge of the disk to saturateit; then, push over the disk with the pipette tiponto the agar so that it lies flat

8 Insert a sterile disk on the negative control plate

in the same manner as above Moisten this diskwith sterile water, and reposition it flat on the agarsurface Be sure to use a fresh Pasteur pipette

9 Place a disk on the unknown plate, and, using the

same procedures, infiltrate it with your unknown,and position it flat on the agar

10 On the fourth plate (optional) deposit a drop of

your unknown from home If the test substancefrom home is crystalline, place a few crystals di-rectly on the top agar of the optional plate in itscenter Liquid substances should be handled insame manner as above

11 Incubate all four plates for 48 hours at 37° C

(Evaluation)

Examine all four plates You should have a nounced halo of revertant colonies around the disk onthe positive control plate and no, or very few, rever-tants on the negative control plate The presence of afew scattered revertants on the negative control plate

pro-is due to spontaneous back mutations, which alwaysoccur Examine the areas beyond the halo to see if youcan detect a faint lawn of bacterial growth

Exercise 76 • Bacterial Mutagenicity and Carcinogenesis: The Ames Test

CAUTION

Since much of the glassware in this experiment tains carcinogens, do not dispose of any of it in theusual manner Your instructor will indicate how thisglassware is to be handled

con-Applications Lab Manual,

Eighth Edition

Medical Microbiology and Immunology

Although many of the exercises up to this point in this manual tain in some way to medical microbiology, they also have applica-tions that are nonmedical The exercises of this unit, however, areprimarily medical or dental in nature

per-Medical (clinical) microbiology is primarily concerned with theisolation and identification of pathogenic organisms Naturally, thetechniques for studying each type of organism are different A com-plete coverage of this field of microbiology is very extensive, en-compassing the Mycobacteriaceae, Brucellaceae, Enterobacte-

riaceae, Corynebacteriaceae, Micrococcaceae, ad infinitum It is

not possible to explore all of these groups in such a short period oftime; however, this course would be incomplete if it did not includesome of the routine procedures that are used in the identification ofsome of the more common pathogens

Exercise 77 in this unit differs from the other 13 exercises in that

it pertains to the spread of disease (epidemiology) rather than tospecific microorganisms Its primary function is to provide an un-derstanding of some of the tools used by public health epidemiol-ogists to determine the sources of infection in the disease trans-mission cycle

Since the most frequently encountered pathogenic bacteria arethe gram-positive pyogenic cocci and the intestinal organisms,Exercises 78, 79, and 80 have been devoted to the study of thosebacteria The exercise that provides the greatest amount of depth

is Exercise 79 (The Streptococci) To provide assistance in the tification of streptococci, it has been necessary to provide supple-mentary information in Appendix E

iden-Four exercises (82, 83, 84, and 85) are related to various cations of the agglutination reaction to serological testing Two ofthese exercises pertain to slide tests and two of them are tubetests It is anticipated that the instructor will select those tests fromthis group that fit time and budget limitations

appli-Exercises 87, 88,and 89 cover some of the basic hematologicaltests that might be included in a microbiology laboratory The lastexercise (90) pertains to an old test that has been revived pertain-ing to caries susceptibility

14

Applications Lab Manual,

Eighth Edition

A Synthetic Epidemic

77

A disease caused by microorganisms that enter the

body and multiply in the tissues at the expense of the

host is said to be an infectious disease Infectious

dis-eases that are transmissible to other persons are

con-sidered to be communicable The transfer of

commu-nicable infectious agents between individuals can be

accomplished by direct contact, such as in

handshak-ing, kisshandshak-ing, and sexual intercourse, or they can be

spread indirectly through food, water, objects,

ani-mals, and so on

Epidemiology is the study of how, when, where,

what, and who are involved in the spread and

distrib-ution of diseases in human populations An

epidemi-ologist is, in a sense, a medical detective who searches

out the sources of infection so that the transmission

cycle can be broken

Whether an epidemic actually exists is

deter-mined by the epidemiologist by comparing the

num-ber of new cases with previous records If the numnum-ber

of newly reported cases in a given period of time in a

specific area is excessive, an epidemic is considered

to be in progress If the disease spreads to one or more

continents, a pandemic is occurring.

In this experiment we will have an opportunity to

approximate, in several ways, the work of the

epi-demiologist Each member of the class will take part

in the spread of a “synthetic infection.” The mode of

transmission will be handshaking For obvious safety

reasons, the agent of transmission will not be a

pathogen

Two different approaches to this experiment are

given: procedures A and B In procedure A a white

powder is used In Procedure B two non-pathogens

(Micrococcus luteus and Serratia marcescens) will

be used The advantage of procedure A is that it can

be completed in one laboratory session Procedure B,

on the other hand, is more realistic in that viable

or-ganisms are used; however, it involves two periods

Your instructor will indicate which procedure is to be

followed

ered the infectious agent The other members will beissued a transmissible agent that is considered nonin-fectious After each student has spread the powder onhis or her hands, all members of the class will engage

in two rounds of handshaking, directed by the structor A record of the handshaking contacts will berecorded on a chart similar to the one on theLaboratory Report After each round of handshaking,the hands will be rubbed on blotting paper so that achemical test can be applied to it to determine thepresence or absence of the infectious agent

in-Once all the data are compiled, an attempt will bemade to determine two things: (1) the original source

of the infection, and (2) who the carriers are The type

of data analysis used in this experiment is similar tothe procedure that an epidemiologist would employ.Proceed as follows:

Materials:

1 numbered container of white powder*

1 piece of white blotting paperspray bottles of “developer solution”*

Preliminaries

1 After assembling your materials, write your nameand unknown number at the top of your sheet ofblotting paper In addition, draw a line down themiddle, top to bottom, and label the left sideROUND 1 and the right side ROUND 2

2 Wash and dry your hands thoroughly

3 Moisten the right hand with water and prepare itwith the agent by thoroughly coating it with thewhite powder, especially on the palm surface.This step is similar to the contamination thatwould occur to one’s hand if it were sneezed intoduring a cold

IMPORTANT: Once the hand has been prepared

do not rest it on the tabletop or allow it to touchany other object

Applications Lab Manual,

Eighth Edition

Round 1

1 On the cue of the instructor, you will begin the

first round of handshaking Your instructor will

inform you when it is your turn to shake hands

with someone You may shake with anyone, but it

is best not to shake your neighbor’s hand Be sure

to use only your treated hand, and avoid

ex-tracurricular glad-handing.

2 In each round of handshaking you will be selected

by the instructor only once for handshaking;

how-ever, due to the randomness of selection by the

handshakers, it is possible that you may be

se-lected as the “shakee” several times

3 After every member of the class has shaken

some-one’s hand, you need to assess just who might

have picked up the “microbe.” To accomplish

this, wipe your fingers and palm of the

contami-nated hand on the left side of your blotting paper

Press fairly hard, but don’t tear the surface

IMPORTANT: Don’t allow your hand to touch

any other object A second round of handshaking

follows

Round 2

1 On the cue of your instructor, shake hands with

another person Avoid contact with any other

ob-jects

2 Once the second handshaking episode is finished,

rub the fingers and palm of the contaminated hand

on the right side of the blotting paper

CAUTION: Keep your contaminated hand off

the left side of the blotting paper

Chemical Identification

1 To determine who has been “infected” we will

now spray the developer solution on the

hand-prints of both rounds One at a time, each student,

with the help of the instructor, will spray his or

her blotting paper with developer solution

2 Color interpretation is as follows:

Blue:—positive for infectious agent

Brown or yellow:—negative

Tabulation of Results

1 Tabulate the results on the chalkboard, using a

table similar to the one on the Laboratory Report

2 Once all results have been recorded, proceed to

determine the originator of the epidemic The

eas-iest way to determine this is to put together a

flowchart of shaking

3 Identify those persons that test positive You will

be working backward with the kind of

informa-tion an epidemiologist has to work with (contactsand infections) Eventually, a pattern will emergethat shows which person started the epidemic

4 Complete the Laboratory Report

In this experiment each student will be given a piece

of hard candy that has had a drop of Micrococcus

lu-teus or Serratia marcescens applied to it Only one

person in the class will receive candy with S.

marcescens, the presumed pathogen All others will

receive M luteus.

After each student has handled the piece of candywith a glove-covered right hand, he or she will shakehands (glove to glove) with another student as di-rected by the instructor A record will be kept of whotakes part in each contact Two rounds of handshakingwill take place After each round, a plate of trypticasesoy agar will be streaked

After incubating the plates, a tabulation will be

made for the presence or absence of S marcescens on

the plates From the data collected, an attempt will bemade to determine two things: (1) the original source

of the infection and (2) who the carriers are The type

of data analysis used in this experiment is similar tothe procedure that an epidemiologist would employ.Proceed as follows:

Materials:

sterile rubber surgical gloves (1 per student)

hard candy contaminated with M luteus hard candy contaminated with S marcescens

sterile swabs (2 per student)TSA plates (1 per student)

Preliminaries

1 Draw a line down the middle of the bottom of aTSA plate, dividing it into two halves Label onehalf ROUND 1 and the other ROUND 2

2 Put a sterile rubber glove on your right hand.Avoid contaminating the palm surface

3 Grasp the piece of candy in your gloved hand,rolling it around the surface of your palm Discardthe candy into a beaker of disinfectant set asidefor disposal You are now ready to do the first-round handshake

A Synthetic Epidemic • Exercise 77

CAUTION

Although the pathogenicity of S marcescens is

con-sidered to be relatively low, avoid allowing any skincontact during this experiment

Applications Lab Manual,

Eighth Edition

Round 1

1 On the cue of your instructor, select someone to

shake hands with You may shake with anyone,

but it is best not to shake hands with your

neighbor

2 In each round of handshaking you will be selected

by the instructor only once for handshaking;

how-ever, due to the randomness of selection by the

handshakers, it is possible that you may be

se-lected as the “shakee” several times The

instruc-tor or a recorder will record the initials of the

shaker and shakee each time

3 After you have shaken someone’s hand, swab the

surface of your palm and transfer the organisms to

the side of your plate designated as ROUND 1

Discard this swab into the appropriate container

for disposal

Round 2

1 Again, on the cue of your instructor, select

some-one at random to shake hands with Be sure not to

contaminate your gloved hand by touching

some-thing else

2 With a fresh swab, swab the palm of your hand

and transfer the organisms to the side of your

plate designated as ROUND 2 Make sure thatyour initials and the initials of the shakee arerecorded by the instructor or recorder

3 Incubate the TSA plate at room temperature for

48 hours

Tabulation and Analysis

1 After 48 hours’ incubation look for typical red S.

marcescens colonies on your Petri plate If such

colonies are present, record them as positive onyour Laboratory Report chart and on the chart onthe chalkboard

2 Fill out the chart on your Laboratory Reportwith all the information from the chart on thechalkboard

3 Identify those persons that test positive Youwill be working backwards with the kind of in-formation an epidemiologist has to work with(contacts and infections) Eventually a patternwill emerge that shows which person started theepidemic

Complete the Laboratory Report for this exercise

Exercise 77 • A Synthetic Epidemic

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Eighth Edition

and Immunology Isolation and Identification Companies, 2001

78

The Staphylococci:

Isolation and Identification

tions (3), and vascular graft infections (1) have beenshown to be due to coagulase-negative staphylo-cocci Numbers in parentheses designate references

at the end of this exercise

Our concern in this exercise will pertain sively to the differentiation of only three species ofstaphylococci If other species are encountered, thestudent may wish to use the API Staph-Ident minia-turized test strip system (Exercise 55)

exclu-In this experiment we will attempt to isolatestaphylococci from (1) the nose, (2) a fomite, and (3)

an “unknown-control.” The unknown-control will be

a mixture containing staphylococci, streptococci, andsome other contaminants If the nasal membranes andfomite prove to be negative, the unknown-control willyield positive results, providing all inoculations andtests are performed correctly

Since S aureus is by far the most significant

pathogen in this group, most of our concern here will

be with this organism It is for this reason that thecharacteristics of only this pathogen will be outlinednext

Staphylococcus aureus cells are 0.8 to 1.0 ␮m indiameter and may occur singly, in pairs, or as clusters

Colonies of S aureus on trypticase soy agar or blood

agar are opaque, 1 to 3 mm in diameter, and yellow,orange, or white They are salt-tolerant, growing well

Often in conjunction with streptococci, the

staphylo-cocci cause abscesses, boils, carbuncles, osteomyelitis,

and fatal septicemias Collectively, the staphylococci

and streptococci are referred to as the pyogenic

(pus-forming) gram-positive cocci Originally isolated from

pus in wounds, the staphylococci were subsequently

demonstrated to be normal inhabitants of the nasal

membranes, the hair follicles, the skin, and the

per-ineum of healthy individuals The fact that 90% of

hos-pital personnel are carriers of staphylococci portends

serious epidemiological problems, especially since

most strains are penicillin-resistant

The staphylococci are gram-positive spherical

bacteria that divide in more than one plane to form

ir-regular clusters of cells They are listed in section 12,

volume 2, of Bergey’s Manual of Systematic

Bacteriology The genus Staphylococcus is grouped

with three other genera in family Micrococcaceae:

SECTION 12 GRAM-POSITIVE COCCI

Although the staphylococci make up a coherent

phylogenetic group, they have very little in common

with the streptococci except for their basic

similar-ities of being gram-positive, non-spore-forming

cocci Note that Bergey’s Manual puts these two

genera into separate families due to their inherent

differences

Of the 19 species of staphylococci listed in

Bergey’s Manual, the most important ones are S

au-reus, S epidermidis, and S saprophyticus The single

most significant characteristic that separates these

species is the ability or inability of these organisms to

coagulate plasma: only S aureus has this ability; the

other two are coagulase-negative

Although S aureus has, historically, been

con-sidered to be the only significant pathogen of the

three, the others do cause infections Some

cere-brospinal fluid infections (2), prosthetic joint

infec-Figure 78.1 Staphylococci

Applications Lab Manual,

Eighth Edition

and Immunology Isolation and Identification Companies, 2001

on media containing 10% sodium chloride Virtually

all strains are coagulase-positive Mannitol is

fer-mented aerobically to produce acid Alpha toxin is

produced that causes a wide zone of clear (beta-type)

hemolysis on blood agar; in rabbits it causes local

necrosis and death

The other two species lack alpha toxin (do not

ex-hibit hemolysis) and are coagulase-negative Mannitol

is fermented to produce acid (no gas) by all strains of

S aureus and most strains of S saprophyticus Table

78.1 lists the principal characteristics that differentiate

these three species of staphylococcus

1 Label the three tubes of m-staphylococcus

broth NOSE, FOMITE, and the number of yourunknown-control

2 Inoculate the appropriate tube of

m-staphylo-coccus broth with one or two loopfuls of yourunknown-control

3 After moistening one of the swabs by immersingpartially into the “nose” tube of broth, swab thenasal membrane just inside your nostril A smallamount of moisture on the swab will enhance thepickup of organisms Place this swab into the

“nose” tube

4 Swab the surface of a fomite with the other swabthat has been similarly moistened and deposit thisswab in the “fomite” tube

The fomite you select may be a coin, drinkingglass, telephone mouthpiece, or any other itemthat you might think of

5 Incubate these tubes of broth for 4 to 24 hours at37° C

(Primary Isolation Procedure)

Two kinds of media will be streaked for primaryisolation: mannitol salt agar and staphylococcusmedium 110

Mannitol salt agar (MSA) contains mannitol,7.5% sodium chloride, and phenol red indicator.The NaCl inhibits organisms other than staphylo-cocci If the mannitol is fermented to produce acid,the phenol red in the medium changes color fromred to yellow

Staphylococcus medium 110 (SM110) also tains NaCl and mannitol, but it lacks phenol red Itsadvantage over MSA is that it favors colony pigmen-

con-tation by different strains of S aureus Since this

medium lacks phenol red, no color change takes place

as mannitol is fermented

Materials:

3 culture tubes from last period

2 Petri plates of MSA

2 Petri plates of SM110

1 Label the bottoms of the MSA and SM110 plates

as shown in figure 78.2 Note that to minimize thenumber of plates required, it will be necessary tomake half-plate inoculations for the nose andfomite The unknown-control will be inoculated

on separate plates

Exercise 78 • The Staphylococci: Isolation and Identification

S aureus S epider- S

Table 78.1 Differentiation of three species of staphylococci

Note: NS ⫽ not significant; S ⫽ sensitive; R ⫽ resistant;

(⫹) ⫽ mostly positive

To determine the incidence of carriers in our

classroom, as well as the incidence of the organism on

common fomites, we will follow the procedure

illus-trated in figure 78.2 Results of class findings will be

tabulated on the chalkboard so that all members of the

class can record data required on the Laboratory

Report The characteristics we will look for in our

iso-lates will be (1) beta-type hemolysis (alpha toxin), (2)

mannitol fermentation, and (3) coagulase production

Organisms found to be positive for these three

char-acteristics will be presumed to be S aureus Final

con-firmation will be made with additional tests Proceed

as follows:

(Specimen Collection)

Note in figure 78.2 that swabs that have been applied

to the nasal membranes and fomites will be placed in

tubes of enrichment medium containing 10% NaCl

(m-staphylococcus broth) Since your

unknown-control will lack a swab, initial inoculations from

this culture will have to be done with a loop

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Eighth Edition

and Immunology Isolation and Identification Companies, 2001

The Staphylococci: Isolation and Identification • Exercise 78

-Figure 78.2 Procedure for presumptive identification of staphylococci

Applications Lab Manual,

Eighth Edition

and Immunology Isolation and Identification Companies, 2001

sterile loop, streak out the organisms on the

re-mainder of the agar on that half of each plate

The swabbed areas will provide massive growth;

the streaked-out areas should yield good colony

isolation

4 Repeat step 3 to inoculate the other half of each

agar plate with the swab from the fomite tube

5 Incubate the plates aerobically at 37° C for 24 to

36 hours

(Plate Evaluations and Coagulase/DNase Tests)

During this period we will perform the following

tasks: (1) evaluate the plates from the previous

pe-riod, (2) inoculate blood agar plates, (3) make

gram-stained slides, and (4) perform coagulase and/or

DNase tests on organisms from selected colonies

Proceed as follows:

Materials:

MSA and SM110 plates from previous period

2 blood agar plates

serological tubes containing 0.5 ml of 1:4 saline

dilution of rabbit or human plasma (one tube

for each isolate)

Petri plates of DNase agar

gram-staining kit

Evaluation of Plates

1 Examine the mannitol salt agar plates Has the

phenol red in the medium surrounding any of the

colonies turned yellow?

If this color change exists, it can be

pre-sumed that you have isolated a strain of S

au-reus Record your results on the Laboratory

Report and chalkboard (Your instructor may

wish to substitute a copy of the chart from the

Laboratory Report to be filled out at the

demon-stration table.)

2 Examine the plates of SM110 The presence of

growth here indicates that the organisms are

salt-tolerant Note color of the colonies (white,

yel-low, or orange)

3 Record your observations of these plates on the

Laboratory Report and chalkboard

2 Select staphylococcus-like colonies from theMSA and SM110 plates from the nose and fomitesfor streaking out on another blood agar plate Usehalf-plate streaking methods, if necessary

3 Incubate the blood agar plates at 37° C for 18 to

24 hours Don’t leave plates in incubator longer

than 24 hours Overincubation will cause blood

degeneration

Coagulase Tests

The fact that 97% of the strains of S aureus have

proven to be coagulase-positive and that the other two

species are always coagulase-negative makes the

co-agulase test an excellent definitive test for confirming

identification of S aureus.

The procedure is simple It involves inoculating asmall tube of plasma with several loopfuls of the or-ganism and incubating it in a 37° C water bath for sev-eral hours If the plasma coagulates, the organism iscoagulase-positive Coagulation may occur in 30

minutes or several hours later Any degree of

coagula-tion, from a loose clot suspended in plasma to a solid immovable clot, is considered to be a positive result, even if it takes 24 hours to occur.

It should be emphasized that this test is valid only for gram-positive, staphylococcus-like bacte- ria, because some gram-negative rods, such as

Pseudomonas, can cause a false-positive reaction.

The mechanism of clotting in such organisms is notdue to coagulase Proceed as follows:

1 Label the plasma tubes NOSE, FOMITE, or KNOWN, depending on which of your plateshave staph-like colonies

UN-2 With a wire loop, inoculate the appropriate tube

of plasma with organisms from one or morecolonies on SM110 or MSA Use several loop-fuls Success is more rapid with a heavy inocula-tion If positive colonies are present on both noseand fomite sides, be sure to inoculate a separatetube for each side

3 Place the tubes in a 37° C water bath

4 Check for solidification of the plasma every 30minutes for the remainder of the period Note infigure 78.3 that solidification may be complete, as

in the lower tube, or show up as a semisolid ball,

as seen in the middle tube

Any cultures that are negative at the end ofthe period will be left in the water bath At 24hours your instructor will remove them from the

Exercise 78 • The Staphylococci: Isolation and Identification